Abused substances produce long-term brain changes including neurotoxicity, through mechanisms that are largely unknown. The candidate neuromodulator nitric oxide (NO) can mediate aspects of NMDA-induced neurotoxicity in vivo and in primary cultured neurons. In this FY, we have extended observations made during the previous fiscal year that show that in situations of excess glutamate stimulation, No mediates cell death in culture and therefore may mediate the cellular destruction observed following stroke, brain injury, neurodegenerative diseases, and severe drug abuse. Mature primary culture neurons were exposed to various drug solutions and the resultant cell death was assayed. NMDA neurotoxicity was found in rat primary cell cultures of cortex, striatum and hippocampus. Studies using inhibitors of NOS and scavengers of NO demonstrated that NO mediates toxicity and that a select population (2-5%) of neurons kills surrounding neurons. Modulating the activity of NOS by inhibiting the cofactor calmodulin, by changing its phosphorylation state or by inhibiting the shuttling of electrons through its flavoproteins modulates NMDA neurotoxicity in the same manner as NOS activity. As NOS activity is decreased so is neurotoxicity. NOS is stimulated by Ca 2+ influx into the cell either by NMDA receptor activation or by the calcium ionophore, A23187. Toxicity due to A23187 can be modulated in the same manner as NMDA toxicity by altering the activity of NOS. Neurons, not microglia, are the source of neurotoxic NO; neurotoxicity in dependent on the presence of NOS-expressing neurons in culture. Guanylate cyclase and cGMP are not involved in neurotoxicity but the superoxide anion is essential.